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PubMed1992

Management of sedimentation in centrifugal counter-current distribution of sperm cells in an aqueous 2-phase system.

It is generally assumed that centrifugal counter-current distribution (CCCD) in aqueous two-phase systems cannot be employed for analyzing or fractioning cell populations, due to large particles of sediment in the system caused by enhanced gravity. The present work was undertaken to find out whether addition of Percoll to a two-phase system would be a useful method to avoid this cell sedimentation. The results obtained show that bull spermatozoa partition as a unique peak in a CCCD using a Dextran T500-poly(ethylene glycol) 6000 system, and that sedimentation takes place significantly in the upper phase during the process. Addition of increasing concentrations of Percoll made this unique peak wider and two different populations of bull spermatozoa were finally obtained when Percoll concentration rose to 13.6%. This management of cell sedimentation in CCCD could be of great interest for analyzing cell heterogeneity, since the shortening of the time required for counter-current distribution should prevent the loss of cell viability during the separation process. Finally, the results obtained suggest that an increase of viscosity rather than of density is the phase feature which has greater influence on managing cell sedimentation in CCCD.

Animals↗
PubMed1990

Settling-time dependence of rat bone marrow cell partition and counter-current distribution in charge-sensitive aqueous two-phase systems. Relationship with the cell partitioning mechanism.

Differences in the settling-time dependence of single and multiple cell partitions have been found between heterogeneous (bone marrow cells) and homogeneous (erythrocytes) populations when using charge-sensitive dextran-poly(ethylene glycol) aqueous two-phase systems. The cell populations were partitioned using both single test-tube experiments and multiple thin-layer counter-current distribution. Lengthening the settling time, to favour phase separation, and decreasing the upper phase volume are more effective in fractionation by the counter-current distribution of heterogeneous cell populations than increasing the interfacial tension, although all three were employed to speed phase settling. On the basis of these results, the original cell partitioning mechanism proposed for non-charge-sensitive systems has been extended to charge-sensitive systems.

Animals↗
PubMed1989

Heterogeneity of a crude synaptosomal preparation, studied by affinity partitioning using hexaethonium-poly(ethylene glycol).

The heterogeneity of a synaptosomal preparation was studied by the use of affinity partitioning in combination with centrifugal counter-current distribution. Hexaethonium-poly(ethyleneglycol) was used as the extracting agent. The fractions were analyzed for: light scattering, protein, choline acetyltransferase, L-glutamate decarboxylase, glutamine synthetase, 2',3'-cyclicnucleotide-3'-phosphohydrolase, acetylcholinesterase and succinate dehydrogenase. The material was fractionated into three main fractions which differed in their content of marker-enzymes.

Animals↗
PubMed1994

Biochemical characterization of human erythrocytes fractionated by counter-current distribution in aqueous polymer two-phase systems.

The fractionation of normal human erythrocytes by counter-current distribution (CCD) in charge-sensitive dextran-polyethylene glycol two-phase systems was confirmed and extended to red blood cells from heterozygous beta-thalassaemic patients. The differences between the distribution profiles of normal (homogeneous) and abnormal (heterogeneous) red blood cells reflect their different surface-charge properties. As suggested by the decline of membrane sialic acid released after neuraminidase treatment and the specific activities of two age-dependent enzymes (membrane acetylcholinesterase and intracellular pyruvate kinase) in the distribution profiles (from the left- to the right-hand side fractions), the fractionation seems to be according to red blood cell age. A constancy of the 2,3-bisphosphoglycerate level was observed in ageing red blood cells.

2,3-Diphosphoglycerate↗
PubMed1978

Portal circulations and their relation to counter-current systems.

We have reviewed the distribution of portal circulations throughout the animal body; they are commoner than is generally supposed. Most portal circulations consist of two serial capillary beds connected by one or more larger vessels. We have called these 'convergent' portal circulations: examples are hepatic portal, placental, hypophysial, renal, ovarian and testicular circulations, as are parts of the lymphatic circulation. A second type of portal circulation, which is less common, consists of two serial capillary beds that are not connected by larger vessels. These we have called 'continuous' portal circulations: adrenal and pancreatic circulations are examples of this type. When a countercurrent concentrating mechanism exists in the body it is always part of the primary or secondary bed of a convergent portal circulation, though some convergent portal circulations are not associated with countercurrent mechanisms.

Adrenal Glands↗
PubMed1981

Separation by counter-current distribution of rat and chicken erythrocytes of different age, and its application to the assay of enzyme activities.

The separation of cells with different ages from erythrocyte populations of adult rats and young or adult chickens have been achieved by counter-current distribution (CCD). A thin-layer CCD apparatus has been employed. Erythrocytes from blood samples taken at different times after 59Fe i.p. injection were separated by CCD. By compilation in a "composite curve" of the hemoglobin and radioactivity CCD profiles obtained for each erythrocyte population, the distribution of cells according to age can be inferred. Young erythrocytes of rats are located at the right part of the CCD curves, while older cells are distributed towards the left. An opposite distribution has been found for erythrocytes from adult or young chickens. As a first attempt for the application of the CCD procedure to the assay of enzyme activities, it was found a decrease in phytase activity as the age of chicken erythrocytes increases and an increase in phosphoglycerate kinase and phosphofructokinase as the age of rat erythrocytes increases.

6-Phytase↗
PubMed1978

Biochemical studies on rat liver Golgi apparatus. III. Subfractionation of fragmented Golgi apparatus by counter-current distribution.

Vesicular fragments of Golgi apparatus, smooth- and rough-surfaced microsomes from rat liver are differently partitioned in aqueous polymer two-phase systems consisting of dextran, polyethylene glycol, and sodium phosphate buffer. At a given polymer concentration, the amount of material partitioned in the top phase increases in the following order: rough microsomes less than smooth microsomes less than Golgi fragments. Counter-current distribution of Golgi fragments in the system consisting of 6.8% (w/w) dextran T500 and 6.8% polyethylene glycol 4,000 results in the separation of the fragments into three fractions; i.e. Fractions I, II, and III. NADH- and NADPH-cytochrome c reductase activities are detected almost exclusively in Fraction I, whereas the activities of galactosyltransferase, acid phosphatase, 5'-nucleotidase, and thiamine pyrophosphatase are maximal in Fraction III and minimal in Fraction I. The distribution of these enzymes suggests that Fraction I is similar to, though not identical with, microsomes, Fraction III resembles plasma membrane and lysosomes, and Fraction II is between the two. It is concluded that NADH- and NADPH-cytochrome c reductases are localized in a restricted region of the Golgi structure and that intra-Golgi differentiation seems to proceed in a discontinuous manner.

Acid Phosphatase↗
PubMed1988

Partitioning behaviour in aqueous two-phase systems and fractionation by counter-current distribution of chick-embryo erythrocytes with numerical resolution of distribution curves.

The partition of chick-embryo and young-chick erythrocytes in dextran-poly-(ethylene glycol) two-phase systems depends on the interfacial tension and electrical potential differences between the phases. Counter-current distribution with charged 5% dextran-poly(ethylene glycol) systems has proved to be an adequate method for the separation of primitive and definitive erythrocytes present in chick embryos when a phase settling time of 20 min is used. The computer-aided numerical resolution of experimental curves has shown the existence of subpopulations which could not have been detected by using conventional methods.

Animals↗
PubMed1991

Mutagenic contaminants in synthetic peptides obtained by an azide coupling.

Hormone-like peptides are, almost by definition, not mutagenic. It was, therefore, unusual to find that some batches of peptides synthesized by azide coupling were mutagenic in the Ames test. One of these peptides, eledoisin, showed mutagenic activity particularly in Salmonella typhimurium TA 1535 without metabolic activation. This activity was independent of the peptide purity determined by HPLC and a dose response relationship was observed at concentrations over the solubility limit of the peptide in the assay medium. We therefore suggested that the mutagenic effect might be due to the presence of chemically undetectable, water-soluble impurities, which could be removed by counter-current distribution. If, however, the same final coupling was carried out by the mixed anhydride procedure, no mutagenic activity was observed. Consequently, we considered that the mutagenicity detected was due to traces of hydrazoic acid salts arising during azide formation in the coupling step. In fact only the product of the coupling reaction between the pivotal intermediates was mutagenic.

Amino Acid Sequence↗
PubMed1985

Demonstration of two erythrocyte populations in young chickens by counter-current distribution of 59Fe-labelled cells in dextran-poly(ethylene glycol) two-phase systems.

The technique of fractionating cells by a counter-current distribution procedure in dextran-poly(ethylene glycol) biphasic systems was modified by increasing the settling time and decreasing the top/bottom two-phase volume ratio. Two sub-populations of 59Fe-labelled erythrocytes, namely definitive medullar cells and primitive embryonic cells, were present in the blood of young chicks until about day 8 of age. The primitive cells were progressively replaced by definitive red cells as age increased.

Aging↗
PubMed1982

Microanalytical partition of rat-liver homogenates by poly(ethylene glycol)-dextran counter-current distribution.

1. The principal organelles from rat liver homogenates were fractionated by counter-current partition between solutions of dextran T50 and poly(ethylene glycol) 6000 in 0.25 M sucrose containing 10 mM sodium phosphate/phosphoric acid buffer, pH 7.4. 2. A small-volume (1.3-ml sample well)counter-current partition apparatus capable of 17 transfers, in which separation of the phases was enhanced by low-speed centrifugation, was used. 3. Variations in distribution of organelles was observed with increasing concentration of dextran and/or poly(ethylene glycol). At a constant polymer composition of 5.0% (w/w) dextran and 4.0% (w/w) poly(ethylene glycol), increasing concentrations of sodium chloride (2.5 mM, 5 mM and 10 mM) also altered the partition of the organelles. 4. Useful analytical resolution of the various organelles was achieved. In particular, separation of endoplasmic reticulum from plasma membrane components was obtained.

Animals↗
PubMed1976

Isolation and identification of a sulfur-containing metabolite of spironolactone from human urine.

In the urine of normal subjects who were given an oral dose of 500 mg spironolactone (3-(3-oxo-7alpha-acetylthio-17beta-hydroxy-4-androsten-17alpha-yl)-propionic acid gamma-lactone; Aldactone) together with 100uCi H-20, 21 spironolactone, a so far unknown major metabolite has been detected by thin layer chromatography. The metabolite then could be isolated by means of counter-current-distribution. According to masspectral and magnetic resonance data, the metabolite has been assigned the structure of 3-(3-oxo-7alpha-methyl sulfonyl-6beta, 17beta-dihydroxy-4-androsten-17alpha-yl)-propionic acid gamma-lactone. By oxidation of the corresponding methylsulfinyl compound - another already known metabolite of spironolactone - with m-chloroperbenzoic acid, a compound has been isolated which proved to be identical with the new metabolite according to TLC, MS and NMR.

Countercurrent Distribution↗
PubMed1985

Affinity partitioning and centrifugal counter-current distribution of membrane-bound opiate receptors using naloxone-poly(ethylene glycol).

Crude synaptic membranes isolated from calf brain cortex were subjected to an aqueous two-phase system and the partition of the various membrane constituents and activities between the phases were studied. These constituents were phosphate, cholesterol and protein. The activities measured were acetyl-cholinesterase, succinate dehydrogenase, 2',3'-cyclicnucleotide-3'-phosphohydrolase and stereospecific opiate-binding. The successful fractionation of the membranes was achieved by the use of an aqueous two-phase system in a counter-current distribution process. A ligand bound to poly(ethylene glycol) with an affinity for opiate receptors was synthesized by reacting 6-aminonaloxone with tresylpoly(ethylene glycol). The ligand-polymer was used to extract membrane-bound opiate receptors into the upper, poly(ethylene glycol)-rich phase. This use of affinity partitioning resulted in membrane fractions with a 3-4 fold higher ability to bind stereospecifically etorphine than the original preparations of synaptic membranes.

Animals↗
PubMed1973

Partition of Salmonella typhimurium in a two-polymer acqueous phase system in relation to liability to phagocytosis.

Analysis of the partition in a two-polymer phase system of Salmonella typhimurium R mutants with different susceptibility to phagocytosis distinguished between the mutants. Close to 80% of the R mutants least resistant to phagocytosis accumulated in the dextran-rich phase, whereas more phagocytosis-resistant mutants showed higher affinity for the interface and the polyethylene glycol-rich top phase. By growing the uridine diphosphate (pyro)-gal-4-epimeraseless mutant LT2-M1 in the presence of d-galactose for different periods of time, two well-defined peaks were obtained in counter-current distribution analysis with an aqueous two-polymer phase system revealing a heterogeneous population in the culture. One peak was located at a site characteristic of R bacteria, the other at the site of S bacteria. As growth proceeded, more bacteria transferred from the R-type to the S-type peak. Within each peak, a gradual increase in resistance to phagocytosis by rabbit polymorphonuclear cells occurred with increasing length of growth in d-galactose.

Countercurrent Distribution↗
PubMed1983

Association of rabbit muscle glycolytic enzymes with filamentous actin. A counter-current distribution study at high ionic strength.

The association between purified glycolytic enzymes and filamentous actin from rabbit muscle has been studied by counter-current distribution. The co-distribution of a glycolytic enzyme and filamentous actin leads to a significant change in the counter-current distribution profile of the enzyme whereas that of actin is unaffected. The changes in the distribution profiles clearly demonstrated that all glycolytic enzymes studied, though to different extents, bind to filamentous actin. The aqueous two-phase system used for the studies contained dextran, poly(ethyleneglycol) and 150 millimolal potassium phosphate buffer, pH 7.0. Since the ionic strength of the two-phase system is determined mainly by the buffer, the glycolytic enzymes are evidently able to associate with filamentous actin, at least in the presence of neutral polymers, at ionic strengths comparable to or higher than those assumed to prevail in vivo.

Actins↗
PubMed1986

Fractionation of bone-marrow cells by counter-current distribution in aqueous polymer two-phase systems. Relation between settling time and the efficiency of separation.

The fractionation of heterogeneous populations of rat and human bone-marrow cells has been studied by counter-current distribution in a charged 5% dextran-4% poly(ethylene glycol) two-phase system. The subfractionation into two broad populations has been achieved at a low top/bottom phase volume ratio by increasing (up to 20 min) the settling time allowed for the phases to be separated after each mixing step. No effect of this parameter on a homogeneous population of erythrocytes has been observed. However, heterogeneous cell separations can be improved by exploiting different phase settling times.

Animals↗
PubMed1982

[Separation of cells of the erythrocyte series of birds and mammals by distribution in biphasic polyethylene glycol-dextran systems].

Experimental conditions for the partition, in dextran (Dx) polyethylene glycol (PEG) biphasic systems, of circulating red cells, reticulocytes and bone marrow cells from adult rats, as well as of erythrocytes from chicken embryos, young animals and adult chickens, have been studied. Once the partition coefficient (K) has been calculated under optimal experimental conditions, a counter-current distribution (CCD) apparatus has been used for the separation of cells in those populations, and the influence of different factors on the distribution of cells has been analyzed. An increase in the distribution efficiency of heterogeneous cells (those from the chicken and bone marrow of rats) is achieved by increasing the sedimentation time during the CCD procedure. The position of cells (previously labelled with 59Fe) along the rotor of the CCD apparatus is related to cells age, and thus allowed us to apply the CCD procedure for the assay of some enzyme activities in the separated cells.

Animals↗
PubMed1989

The use of aqueous two-phase systems to concentrate and purify bovine leukemia virus outer envelope protein gp51.

Enzootic bovine leucosis is a chronic lymphoproliferative disease of cattle. The causative agent, bovine leukemia virus (BLV), is related to the human retroviruses HTLV-I and -II. The external env-protein of BLV, a glycoprotein of 51 kDa, carries neutralizing epitopes and should be an essential component in a vaccine against the virus. Problems have been encountered with the concentration and purification of intact virions of BLV and other retroviruses. During centrifugation procedures the external env-proteins are to a great extent detached and consequently poorly recovered with the virion particles. Therefore, other methods are sought to obtain a high yield of the external glycoproteins. The use of two-phase systems based on water soluble polymers is described for the extraction of BLV-gp51 from culture medium. Several polymer systems were tested and the results showed that some were attractive for large scale application. The classical combination dextran-polyethylene glycol gave promising results; a partition coefficient of about 0.02 was obtained for the distribution of the gp51 between the top and combined inter- and bottom phases. In a single extraction step it was possible to obtain 45% of the glycoprotein in a small volume bottom phase and at the same time about 15-fold purified. That should be compared with a recovery of less than 20% with the conventional centrifugation procedures. It is concluded that extraction in phase systems based on water soluble polymers is a methodology well suited for the concentration and purification of BLV-gp51.

Animals↗